Project 2 focuses on the biology of cardiac stem cells (CSCs). Such resident stem cells within the heart were unrecognized until 2003, but now are believed to form a reservoir for cardiac self-repair. CSCs appear to be more cardiogenic than other stem cells. While they can differentiate into the three relevant cardiac lineages (myocardium, endothelium and vascular smooth muscle), they do not lead to undesired teratoma formation. A central hypothesis of this proposal is the notion that transplantation of CSCs will improve post-ischemic cardiac function; this has been shown in animal models, but not yet in humans. A major technical innovation drives our interest in CSCs: We have now developed methods to isolate and grow CSCs from human endomyocardial biopsy specimens, opening up the prospect for autologous cell therapy for myocardial regeneration. This project lays the foundation for the use of CSCs and their progeny for human therapeutics in Project 3. In addition, we will study in detail the process of CSC differentiation. Here we are fortunate to exploit the finding that, when grown in suspension culture, CSCs readily and spontaneously produce round multicellular structures which express molecular myocardial markers and which can become excitable and contractile. These "cardiospheres" (CSps) are not only interesting biologically but may also have certain advantages to CSCs in therapeutics. Using both human and porcine sources, we will develop improved GMP-compliant methods for isolating and growing CSCs; study the process of differentiation of CSCs into CSps and cardiomyocytes; perform electrophysiological, genomic and proteomic analysis of the cells at various stages of differentiation; assess the arrhythmic risk of CSC and CSp transplantation in an in vitro model; and explore genetic modification as a strategy to enhance engraftment and improve electromechanical function after CSC transplantation in the post-Mi pig model. The knowledge to be gained from this project promises to open up new avenues for effectively treating heart disease.